Transcriptomic Evidence for the Expression of Horizontally

Transcriptomic Evidence for the Expression of HorizontallyTransferred Algal Nuclear Genes in the Photosynthetic SeaSlug, Elysia chlorotica

Sidney K. Pierce,�,*,1,2 Xiaodong Fang,�,3 Julie A. Schwartz,1 Xuanting Jiang,3 Wei Zhao,3

Nicholas E. Curtis,1 Kevin M. Kocot,4 Bicheng Yang,3 and Jian Wang*,3,4

1Department of Integrative Biology, University of South Florida2Department of Biology, University of Maryland3BGI-Shenzhen, Shenzhen, China4Department of Biological Sciences, Auburn University

�These authors contributed equally to this work.

*Corresponding author: E-mail: [email protected]; [email protected].

Associate editor: Charles Delwiche

Abstract

Analysis of the transcriptome of the kleptoplastic sea slug, Elysia chlorotica, has revealed the presence of at least 101chloroplast-encoded gene sequences and 111 transcripts matching 52 nuclear-encoded genes from the chloroplast donor,Vaucheria litorea. These data clearly show that the symbiotic chloroplasts are translationally active and, of even moreinterest, that a variety of functional algal genes have been transferred into the slug genome, as has been suggested byearlier indirect experiments. Both the chloroplast- and nuclear-encoded sequences were rare within the E. chloroticatranscriptome, suggesting that their copy numbers and synthesis rates are low, and required both a large amount ofsequence data and native algal sequences to find. These results show that the symbiotic chloroplasts residing inside thehost molluscan cell are maintained by an interaction of both organellar and host biochemistry directed by the presence oftransferred genes.

Key words: transcriptome, HGT, horizontal gene transfer, chloroplast symbiosis, kleptoplasty, Elysia chlorotica,Vaucheria litorea.

IntroductionCertain cells lining the digestive tubules of several species ofsacoglossan, opisthobranch, sea slugs are able to sequesterchloroplasts from their algal food. The plastids remainintact inside the digestive cells for some length of time, de-pending on the species involved (Pierce and Curtis 2012).Also, in several of these slug species, the captured plastids,often called kleptoplasts, are capable of photosynthesis,and in a few of the species, photosynthesis continuesalmost unabated for as long as a year in the completeabsence of any contact with the algal source of thechloroplasts (Pierce and Curtis 2012).

Most detailed information about various aspects of themechanism of long-term maintenance of the chloroplastsymbiosis has come from work on Elysia chlorotica (Gould),where the sequestered chloroplasts come from the chro-mophytic alga, Vaucheria litorea (C. Agardh). Once thisslug sequesters the chloroplasts, it can continue to pho-tosynthesize for 10–12 months in the absence of any algalfood. Several chloroplast proteins and chlorophyll a aresynthesized during that starvation period, and polymerasechain reaction (PCR) experiments have demonstrated thepresence of at least 11 algal nuclear genes, all involvedin photosynthesis, in E. chlorotica adult and veliger larvalgenomic DNA as well as in adult slug RNA (Pierce et al.

2007, 2009; Rumpho et al. 2008, 2009; Schwartz et al.2010). All of these results show that translationally com-petent algal nuclear genes are present in the slug andthat plastid protein and pigment turnover, necessaryfor sustained photosynthesis, is taking place in the slugcell, supported by horizontal gene transfer (HGT) be-tween the two multicellular species. In addition, a varietyof chloroplast-encoded chloroplast proteins are also syn-thesized while the plastid resides inside the E. chloroticadigestive cell (Mujer et al. 1996; Green et al. 2000). How-ever, recent partial analyses of the transcriptomes of twoother slug species, E. timida and Plakobranchus ocellatus,failed to find any transcriptome sequence reads corre-sponding to algal nuclear genes, which lead to the provoc-ative conclusion that, in spite of the entire foregoing, HGThas not occurred between slug and algae and that ‘‘saco-glossan are not, in genetic terms, what they eat’’ (Wageleet al. 2011). Perhaps not, at least in the case of the twospecies investigated by Wagele et al. (2011), but severalaspects of this study seem to make such a broad conclusionpremature. For instance, the data set represented only a smallfraction of the transcriptome. Also, the RNA came from justa few specimens and was extracted from whole animals, al-though only a small fraction of cells contain chloroplasts. Inaddition, Wagele et al. (2011) assumed that expression levels

© The Author 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, pleasee-mail: [email protected]

Mol. Biol. Evol. 29(6):1545–1556. 2012 doi:10.1093/molbev/msr316 Advance Access publication December 23, 2011 1545

Research

articleD

ownloaded from

https://academic.oup.com

/mbe/article/29/6/1545/996402 by guest on 16 D

ecember 2021

ofalgal genes in the slugcellswouldbeequivalent to transcrip-tion rates in the algae even though expression levels of at leasttwo nuclear-encoded genes for plastid-targeted proteins aremuch lower in E. chlorotica than in its food alga (Soule 2009).Thus, generalizing negative results obtained from problem-atical data to the rest of the species of kleptoplastic slugs,especially in thepresenceof the large amountof biochemicaldata, includingPCR, and showing thepresenceof transferredgenes, seem inappropriate.

However, another recent report failed to find any evi-dence for transferred algal nuclear genes in the transcrip-tome of E. chlorotica, although 19 chloroplast-encodedgene sequences emerged from the analysis (Pelletreauet al. 2011). Unfortunately, while this study investigatedE. chlorotica, the transcriptome sequencing yielded,20% (14,000) of the contigs of the P. ocellatus transcrip-tome and was not compared with V. litorea native sequen-ces. Thus, the lack of both sequencing depth and databasevoids suggests that rare transcripts could have been easilymissed. Indeed, a conclusion of this analysis was that ‘‘moreexhaustive sequencing may be required’’ to adequatelytest for the presence of transferred genes in E. chlorotica(Pelletreau et al. 2011).

In spite of the issues with these negative studies(Pelletreau et al. 2011; Wagele et al. 2011), both nonethelesspoint to the likelihood that if transferred algal nucleargenes are present in sacoglossan slugs, they will be of verylow copy number, their expression will be low, and knowl-edge of the native algal sequence will facilitate, perhapseven be required for, the annotation. Therefore, insteadof assuming that a large number of transcripts for nuclear-encoded and plastid-encoded proteins would be present inthe slug cell, we have hypothesized that such would be ex-ceedingly rare in the transcriptome and have done our ownanalysis of E. chlorotica creating large amounts of Illumina-generated sequence data. In addition, to facilitate an accu-rate annotation, we have sequenced the genome of V. litoreaas well as the algal transcriptome, to provide a databaseof native algal transcript sequences. Our E. chlorotica tran-scriptome data contain a variety, albeit rare, of chloroplast-encoded transcripts and, in addition, rarer still, at least 111reads that match 52 algal nuclear-encoded sequences, in-cluding one that matches exactly an algal nuclear sequencefound by PCR in E. chlorotica genomic DNA and cDNA in theearlier studies.

Materials and Methods

Animals and AlgaeSpecimens of E. chlorotica were collected from a salt marshnear Menemsha onMartha’s Vineyard, MA, and shipped toTampa, FL. The sea slugs were placed into aerated aquariacontaining artificial seawater made from Instant Oceansalts dissolved in sterilized deionized water at 1,000 mOsm.The aquaria were kept at 10 �C in a cold box equipped withfluorescent lights set on a 14:10 light–dark cycle. The slugswere starved for at least 2 months under the foregoingconditions before use in experiments.

Filaments of V. litorea used in the experiments camefrom a culture that has beenmaintained in our lab for morethan 10 years. The initial filament used to establish the cul-ture came from the same marsh that provided the slugs.The V. litorea filaments are grown in a modified F/2enriched seawater at 250 mOsm in an incubator at20 �C, illuminated by fluorescent lights on a 14:10 light–dark cycle (Pierce et al. 1996). Since the original culturewas established from a single filament and has only grownvegetatively, the filaments used here are clonal.

Extraction of Genomic DNA from V. litoreaGenomic DNA was extracted from 10 g of algal filamentsusing a protocol modified from Al-Samarrai and Schmid(2000). The filaments were rinsed with fresh culture media,blotted to remove excess liquid, frozen in liquid nitrogen,and ground to a powder with a precooled mortar andpestle. Lysis buffer (40 mM Tris–acetate, 20 mM sodiumacetate, 1 mM ethylenediaminetetraacetic acid, and 1%sodium dodecyl sulfate, pH 7.0) was added to the frozenalgal powder and extracted for 10 min at room tempera-ture (RT) while rotating. To facilitate the precipitation ofproteins and polysaccharides, 5 M NaCl was added to eachtube (2:5 v/v), the tubes were vortexed and then centri-fuged at 12,000� g at 4 �C. The supernatant was extractedwith phenol/chloroform (1:1 v/v) and spun at 10,000 � g4 �C. The aqueous phase was extracted with chloroform(1:1 v/v) and centrifuged again at 10,000 � g 4 �C. DNAwas precipitated from the aqueous phase by adding isopro-panol (1:1 v/v) and spun at 10,000 � g 4�C to pellet thenucleic acids. The precipitated DNA was resuspended inlysis buffer containing 100 lg/ml RNase A (Qiagen, Valencia,CA) and rotated for 30 min at RT. Following thatincubation, the DNA was run through the purificationprocess a second time. The final precipitated DNA waswashed twice with 75% ethanol, air dried, resuspended in nu-clease-free water, quantified spectrophotometrically at 260nm, and then express shipped to BGI in Hong Kong, China.

Algal TranscriptomeTwo V. litorea transcriptome data sets were used in ouranalysis. One set was produced earlier by 454 sequencing(Schwartz et al. 2010) and the second set using the Illuminaplatform as described below. The 454 data are referred to asEST and the Illumina data as RNA-seq, hereafter.

Extraction of RNA from V. litoreaTotal RNA was isolated from about 1 gm of algal filamentsafter 5 h of exposure to light. The filaments were groundinto a frozen powder as described above, RNA was ex-tracted using the RNeasy Plant mini kit (Qiagen, Valencia,CA) following the manufacturer’s instructions, quantifiedspectrophotometrically at 260 nm, placed on dry ice,and express shipped immediately to BGI in Hong Kong, CN.

Extraction of RNA from E. chloroticaTotal RNA was isolated from .2-month starved slugs af-ter 8 h of light exposure by homogenization in Trizol Re-agent (Invitrogen, Carlsbad, CA). The homogenate was

Pierce et al. · doi:10.1093/molbev/msr316 MBE

1546

Dow

nloaded from https://academ

ic.oup.com/m

be/article/29/6/1545/996402 by guest on 16 Decem

ber 2021

centrifuged at 12,000 � g at 4 �C to remove cellular debris.The supernatant was extracted with chloroform (1:6 v/v)and centrifuged at 10,000� g at 4 �C. RNAwas precipitatedfrom the aqueous phase by adding isopropanol (1:4 v/v) fol-lowed by 0.8 M Na3C6H5O7/1.2 M NaCl solution (1:4 v/v)and was spun at 12,000 � g at 4 �C. The RNA pellet waswashed twice with 75% ethanol, air dried, and resuspendedin nuclease-free water. The RNA in this final solution wasquantified spectrophotometrically and express shipped toBGI in Hong Kong, CN on dry ice.

Algal Genome Sequencing and AssemblyThe genome of V. litorea was sequenced (Illumina HiSeq2,000 platform) using a whole genome shotgun strategy.To reduce bias, eight paired-end sequencing libraries wereconstructed,with various insert sizes (350bp, 400bp, 800bp,2 kbp, 5 kbp, 10 kbp, and 20 kbp), and sequenced.

Genome AssemblyIn total, 8 Gb, or 86-fold coverage, of high-quality reads wereused in the assembly. The algal genome was assembled usingSOAPdenovo (Li, Zhu, et al. 2010) software (http://soap.genomics.org.cn), using the same procedures described forassembly of the giant Panda genome (Li, Fan, et al. 2010).

A total of 7.9 Gb (or 85.9X) data were retained for as-sembly. All high-quality paired-end reads were aligned intocontigs for scaffold building. This paired-end informationwas subsequently used to link contigs into scaffolds,step-by-step, from short insert sizes to long insert sizes.About 3.9 Gb (or 42.4X) data were used to build contigsfor the algal genome, and all the high-quality data wereused to build scaffolds. Some intrascaffold gaps were filledby local assembly using the reads in a read pair, where oneend uniquely aligned to a contig while the other end waslocated within a gap. The final total contig size and N50were 83.6 Mb and 59.6 Kb, respectively. The total scaffoldsize and N50 were 93.2 Mb and 333.3 Kb, respectively.More than 97% of long ESTs (.500 bp) mapped to thealgal genome, which indicated the high quality of the ge-nome in the transcribed regions.

Gene Annotation Pipeline and Evaluation of GeneQualitySince there is only scant V. litorea sequence informationin the public databases, we used both homology basedand de novo methods to localize gene sequences in thealgal genomic data incorporating reads from both theRNA-seq and EST data. To identify homologous genes,protein sequences from algal species that were availablein NCBI (http://www.ncbi.nlm.nih.gov/), including Ectocarpussiliculosus, Phaeodactylum tricornutum, Micromonas sp.,M. pusilla, Ostreococcus lucimarinus, O. tauri, and Volvoxcarteri, were mapped to the V. litorea genome using TBlastN(Kent 2002). Then, homologous genome sequences werealigned against the matching proteins, using GeneWise(Birney et al. 2004) to define gene models. For de novodiscovery of coding genes, AUGUSTUS (Stanke andWaack 2003), GlimmerHMM, and SNAP were used with

appropriate parameters. ESTs were mapped to the ge-nome with BLAST and assembled to genes with PASA.RNA-seq were mapped to the genome using TopHat(http://tophat.cbcb.umd.edu/), and transcriptome-basedgene structures were obtained with Cufflinks (http://cufflinks.cbcb.umd.edu/). Finally, the homology-basedde novo derived EST prediction and transcript gene setswere merged to form a comprehensive and nonredundantreference gene set using GLEAN (http://sourceforge.net/projects/glean-gene/), removing all the genes which hadonly de novo method support. This procedure produceda reference set of 17,988 V. litorea genomic coding genes.

Transcriptome Sequencing and AssemblyTo prepare the slug and algal transcriptomes for sequenc-ing, poly (A)þ RNA was enriched from the total RNA ofeach species, sheered into fragments, and cDNA wassynthesized by reverse transcription. The cDNA from eachspecies was then sequenced using standard high through-put techniques (Illumina HiSeq2000). All high-quality readswere assembled into contigs longer than 100 bp usingSOAPdenovo software (Li et al. 2010). Contigs were linkedinto scaffolds by mapping reads back to contigs andcombining paired-end information.

Sequence AnalysisAlignment and annotation of the E. chlorotica transcrip-tome sequences were done using the V. litorea coding se-quence database as a reference source (fig. 1) utilizing MPIBLAST 1.5.0 software (Darling et al. 2003; Lin et al. 2005), bymeans of the University of South Florida’s 120 node com-puter cluster platform consisting of dual Intel Xeon X5460Quad Core processors each with 16 GB of memory. Briefly,the slug contigs, scaffolds, and raw reads from the tran-scriptome data were formatted as databases and then com-pared with all 17,988 V. litorea coding sequences using theBlastN algorithm (Altschul et al. 1990) with a cutoff of 1 �10�10. Slug transcripts with significant hits were aligned tothe corresponding V. litorea reference sequence using theClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) sequence alignment program todetermine the location of the matching slug transcripts inthe coding sequence. The corresponding V. litoreamatch-ing sequences were then analyzed using the BlastX algo-rithm (Altschul et al. 1990) to determine if they werenuclear- or chloroplast-encoded in origin by comparisonwith the V. litorea chloroplast genome database in NCBI.Last, slug transcripts matching to algal nuclear-encodedsequences were compared with the assembled slug andalgal transcriptomes using BlastN to determine if the se-quences were located in the contig or raw-read data sets.

Results

Chloroplast-Encoded Transcripts in E. chloroticaOur sequencing runs of the cDNAmade from the E. chloroticatranscriptome produced 98,238,204 reads, which assembledinto 459,299 contigs and 378,851 scaffolds (table 1).

Algal Nuclear Genes in Sea Slug Transcriptome · doi:10.1093/molbev/msr316 MBE

1547

Dow


ic.oup.com/m


ber 2021

http://soap.genomics.org.cn

http://soap.genomics.org.cn

http://www.ncbi.nlm.nih.gov/

http://tophat.cbcb.umd.edu/

http://cufflinks.cbcb.umd.edu/

http://cufflinks.cbcb.umd.edu/

http://sourceforge.net/projects/glean-gene/

http://sourceforge.net/projects/glean-gene/

http://www.ebi.ac.uk/Tools/msa/clustalw2/

http://www.ebi.ac.uk/Tools/msa/clustalw2/

Although relatively rare in number among the contigs,a functionally diverse set of chloroplast-encoded sequenceswere present in the E. chlorotica transcriptome (table 2).One hundred one chloroplast-encoded protein codinggenes were identified in the slug transcriptome sequencedata that matched sequences in both the V. litorea ESTor RNA-seq data, of which 60 were located among the con-tigs and the remaining 41 were present in the raw reads,and also matched sequences in the V. litorea chloroplastgenome (table 2). Transcript sequences coding for proteinsin Photosystem I and II reaction centers, both subunits ofRuBisCO, cytochrome, chlorophyll and ATP synthesis, andRNA polymerase were all discovered (table 2). In addition,matching transcripts associated with protein synthesis andtrafficking were present in the slug and algal transcrip-tomes as well as the V. litorea chloroplast genome data(table 2). An additional 36 sequences, which mapped toa variety of ribosomal protein subunits, matched betweenthe slug and algal transcriptomes as well as with the chlo-roplast genome sequences (table 2). Most of the matchingsequences in the chloroplast-encoded data set were 100%identical, both between slug and algal transcripts as wellas compared with the chloroplast genome, with the excep-tions containing either nonassigned or miscalled bases.

Nuclear-Encoded Algal Sequences in the SlugTranscriptomeAlthough much less common in the transcriptome datathan the chloroplast-encoded transcripts, and not presentin any of the contigs, 111 sequences from the E. chloroticatranscriptome database matched V. litorea reference genesequences, representing 52 putative nuclear-encoded V. litoreagenes (table 3). Among the 111 slug transcripts, 88 were 100%identical, 15 contained 1 bp difference, 4 contained 2 bp

difference, 2 contained 3 bp differences, 1 contained 4 bpdifferences, and 1 contained 6 bp differences to alignedportions of the V. litorea genome coding sequences. Allof the 52 V. litorea genomic coding sequences containedat least two and as many as four nonoverlapping slug tran-scriptome sequences (fig. 2 and table 3; supplementary ma-terial online). So even though the raw reads were only90 bp in length, the match within each V. litorea sequencewas at least 180 bp to as much as 360 bp. Furthermore,in addition to matching the algal coding sequences, 106 ofthe E. chlorotica transcript sequences were also present inthe V. litorea transcriptome (table 3). Twenty-seven of thetranscript sets matching V. litorea genomic coding sequenceswere homologous to genes involved in photosynthesis,carbon fixation, carbohydrate metabolism, thylakoid struc-ture, chaperone activity, and other unique chloroplast pro-cesses. Nineteen V. litorea gene sequences were annotatedas hypothetical or unknown proteins, mostly within theE. siliculosus genome data or other algal species, and the finalsix sequences returned no Blast hit, but all were present inthe slug transcriptome, the V. litorea genomic sequencesand, all but five, in the V. litorea transcriptome (table 3).

As usual, the possibility of contamination of E. chloroticamRNA with algal material in this kind of study is ofconcern, especially considering the rarity of the matchingtranscripts identified in these data. However, the RNA wasextracted from animals that were starved for at least 2months, to ensure that the gut was clear of any algal food.Our previous PCR-based studies with DNA or cDNA ex-tracted from similarly starved slugs have produced negativeresults for nuclear-encoded, non–chloroplast-targeted se-quences for V. litorea ‘‘ITS’’ regions as well as ‘‘SPDS’’ (Pierceet al. 2007, 2009, Schwartz et al. 2010). In addition, the algalnuclear sequences that were found here in the slug

Table 1. Summary of the Elysia chlorotica Transcriptome Sequence Data.

Sequences (n) Base Pairs (Mbp) Mean Length (bp) N50 (bp)

Raw reads 98,238,204 8,841.4 90Contigs (‡100 bp) 459,299 80.7 175 180Scaffold sequences (‡100 bp) 378,851 86.5 228 254

FIG. 1. Flow chart of Elysia chlorotica and Vaucheria litorea transcriptome analyses.


1548

Dow


ic.oup.com/m


ber 2021

http://www.mbe.oxfordjournals.org/lookup/suppl/doi:10.1093/molbev/msr316/-/DC1


Table 2. List of the Chloroplast-Encoded Transcripts in the E. chlorotica Transcriptome and the Corresponding Sequences in the V. litoreaTranscriptome and Chloroplast Genome.

Protein Category Protein Name

Numberof Slug

TranscriptRead Matchesa

Assembledin Slug

Contigs (C)or Raw

Reads (R)

E. chloroticaTranscriptomeSequences

V. litoreaTranscriptome

MatchingSequenceAccessionNumber

V. litoreaChloroplastGenomeMatch

AccessionNumber

(YP0002327xxx.1)

Photosystem Ireaction center

Subunit II 5 R EC Illumina readsb JP709230 575Subunit IV 8 R EC Illumina reads JP709231 498Subunit VII 22 C JP709299c JP709232 573Subunit XI 24 C JP709300 JP709233 559PSI assembly

related protein 4 R EC Illumina reads JP709234 542Plastocyanin-binding

subunit III 12 C JP709301 JP709235 477Photosystem assembly

protein Ycf3 2 R EC Illumina reads E52OB4B01EZIRCd 461Apoprotein A1 568 C JP709302 JP709236 590P700 apoprotein A2 911 C JP709303 JP709236 591

Photosystem IIreaction center

D1 4234 C JP709304 E52OB4B02FP4GF 545D2 97 C JP709305 JP709237 479CP43 apoprotein 131 C JP709306 JP709237 480CP47 chlorophyll

apoprotein 97 C JP709307 JP709238 500Protein T 1 R EC Illumina reads JP709238 501Protein N 1 R EC Illumina reads JP709238 502PSII phosphoprotein 3 R EC Illumina reads No match 503Protein E 6 R EC Illumina reads JP709239 562Protein J 4 R EC Illumina reads No match 565

RuBisCO Small subunit 20 C JP709308 JP709240 543Large subunit 28 R EC Illumina reads JP709241 544

Cytochromesynthesis

Apocytochrome f 57 C JP709309 JP709242 462Cytochrome b6 36 C JP709310 JP709243 486Cytochrome b6/f

complex subunit 4 17 C JP709310 JP709243 487Cytochrome b6/f

complex petM subunit 3 R EC Illumina reads No match 578Cytochrome c553 492 C JP709311 E52OB4B02GOVIB 555Cytochrome c550 oxygen

evolving complex component 178 C JP709312 JP709244 556Ferredoxin 23 R EC Illumina reads JP709245 504Ferredoxin-thioredoxin

reductase catalytic chain 57 C JP709313 JP709246 567Protein involved in

cytochrome c biogenesis 17 R EC Illumina reads JP709247 548Thiamin biosynthesis protein 16 R EC Illumina reads JP709248 572

Chlorophyllsynthesis

Protochlorophyllidereductase ChlB subunit 16 R EC Illumina reads JP709249 464

Protochlorophyllidereductase ATP bindingsubunit (chlI) 181 C JP709314 JP709250 455

Protochlorophyllidereductase ATP bindingsubunit (chlL) 10 R EC Illumina reads JP709251 547

Protochlorophyllidereductase ChlN chain 8 R EC Illumina reads JP709252 546

ATP synthesis orbinding

CfxQ-like protein 31 C JP709315 JP709253 549ATP synthase CF1 subunit b 272 C JP709316 JP709254 459ATP synthase CF1 a subunit 26 C JP709317 JP709255 465ATP synthase CF0 subunit III 12 C JP709318 JP709256 469ATP synthase CF0 subunit IV 16 C JP709319 JP709257 470ABC ATPase 6 R EC Illumina reads No match 541

RNA polymerase a chain 6 R EC Illumina reads JP709258 512b$ subunit 10 R EC Illumina reads No match 473b# subunit 116 C JP709320 JP709259 474b subunit 567 C JP709321 JP709260 475


1549

Dow


ic.oup.com/m


ber 2021

Table 2Continued


Numberof Slug


Assembledin Slug

Contigs (C)or Raw

Reads (R)





AccessionNumber

(YP0002327xxx.1)

Ribosomal L1 1 R EC Illumina reads No match 496L2 179 C JP709322 JP709261 530L3 7 R EC Illumina reads JP709262 533L4 43 C JP709323 JP709263 532L5 15 R EC Illumina reads JP709264 521L6 294 C JP709324 JP709265 519L9 4 R EC Illumina reads No match 493L11 8 C JP709325 JP709266 495L12 2 R EC Illumina reads JP709231 497L13 18 C JP709326 JP709267 511L14 88 C JP709327 JP709268 523L16 117 C JP709328 E52OB4B01BJN0V 526L18 106 C JP709324 E52OB4B01AMWPF 518L19 2 R EC Illumina reads No match 551L20 3 R EC Illumina reads No match 536L21 1 C JP709329 JP709269 588L22 47 C JP709330 JP709270 528L24 7 C JP709327 E52OB4B01DUAI4 522L27 18 C JP709329 JP709269 587L31 14 C JP709331 No match 509L32 1 R EC Illumina reads No match 585L33 1 R EC Illumina reads JP709271 456S2 8 R EC Illumina reads No match 472S3 56 C JP709328 JP709272 527S4 10 C JP709332 JP709273 485S5 556 C JP709333 JP709274 517S7 71 C JP709334 JP709275 507S8 27 C JP709324 JP709264 520S9 18 C JP709335 JP709267 510S11 32 C JP709336 JP709276 513S12 224 C JP709331 JP709277 508S13 341 C JP709337 JP709276 514S14 35 C JP709338 JP709278 592S16 6 R EC Illumina reads JP709279 483S18 2 R EC Illumina reads JP709271 457S19 2 R EC Illumina reads No match 529

Hypothetical YCF3 13 C JP709339 JP709280 458YCF24 24 C JP709340 JP709281 540YCF66 2 R EC Illumina reads JP709282 557Conserved chloroplastprotein 21 C JP709341 JP709283 553

Putative cell divisionprotein FtsH 76 C JP709342 JP709284 499

Others Hsp70-type chaperone 922 C JP709343 JP709285 53460 kDa chaperonin 292 C JP709344 JP709286 574Elongation factor Ts 2 R EC Illumina reads JP709287 471CAB/ELIP/HLIP-likeprotein 2 R EC Illumina reads No match 481

Acetohydroxy acidsynthase large subunit 37 C JP709345 JP709288 489

DNA-replication helicase 15 C JP709346 JP709289 494Translation elongationfactor Tu 404 C JP709347 JP709275 506

Preprotein-translocasesubunit Y 1 R EC Illumina reads E52OB4B02G87QP 516

Preprotein-translocasesubunit 9 C JP709348 JP709290 583

Acetolactate synthasesmall subunit 65 C JP709349 JP709291 535


1550

Dow


ic.oup.com/m


ber 2021

transcripts, corresponded mostly to chloroplast-targetedproteins involved in photosynthesis, or genes involved inprotein processing and chaperone activity. If algal RNA con-tamination was present in the slug material, we would havelikely identified highly expressed, non–photosynthesis-related genes in the data set. But, none were found.

DiscussionThe transcriptome of E. chlorotica contains a variety ofdiverse, although relatively rare, transcripts not only ofV. litorea, chloroplast-encoded, origin, but also of algalnuclear-encoded origin. Clearly, the symbiotic plastidgenome is transcriptionally active within the host celland is producing transcripts for a variety of chloroplast pro-teins. More importantly, we have detected the presence ofseveral transcripts for nuclear-encoded algal proteins inthe slug RNA. While some of the nuclear-encoded readsare for products of presently unknown function, most ofthe annotatable reads code for proteins involved in thesustenance of photosynthesis. Two reads, in particular,correspond to prk, which we (Schwartz et al. 2010) andothers (Rumpho et al. 2009) found in slug genomicDNA and cDNA with PCR experiments. More than 60 algalnuclear-encoded genes have now been demonstrated inE. chlorotica DNA and RNA, either by PCR (Pierce et al.2007, 2009; Rumpho et al. 2009; Schwartz et al. 2010) orby our transcriptome sequencing reported here.

Among the chloroplast-encoded transcripts in theE. chlorotica transcriptome are several that code forcomponents of both PSI and PSII reaction centers, in-cluding D1, D2, and CP43, whose months-long synthesisby the slug had been demonstrated by immunoblot ex-periments (Mujer et al. 1996; Green et al. 2000). Similarly,the presence of chloroplast-encoded transcripts for cytF and both the large and small subunits of RuBisCO verifythe long-term synthesis of those proteins within the

E. chlorotica cell (Green et al. 2000). Also similarly, thepresence of transcripts for a plastid-encoded subunitof ChlI in the chlorophyll synthesis pathway confirms

Table 2Continued


Numberof Slug


Assembledin Slug

Contigs (C)or Raw

Reads (R)





AccessionNumber

(YP0002327xxx.1)

Thiol-specific antioxidantprotein 13 R EC Illumina reads No match 538

Isoflavone reductase 9 R EC Illumina reads JP709292 539Caseinolytic-like Clp

protease 220 C JP709350 JP709293 552Acyl carrier protein

precursor 2 R EC Illumina reads JP709294 589Orf 32 2 R EC Illumina reads No match 586Unidentified reading

frame 1 4 R EC Illumina reads JP709295 463

a Cutoff 5 e�30.b ‘‘EC Illumina reads’’ can be located using NCBI Study accession number SRP009263.2 and are all listed in supplementary table 2, Supplementary Material online.c Sequence identifiers ‘‘JPxxxxxx’’ are NCBI database accession numbers.d All singleton reads in this column labeled ‘‘E52OB4B0xxxxxx’’ are included in Sequence Read Archive Study number SRP009267.2.

FIG. 2. The location of four raw reads (highlighted in gray) in theElysia chlorotica transcriptome within the Vaucheria litorea genomictransketolase coding region sequence (NCBI accession numberJQ062398). All the reads have a 90 bp match with their respectiveregions except for the third one (bp 1857–1946), which has a singlebp difference marked in bold.


1551

Dow


ic.oup.com/m


ber 2021



Table 3. List of Algal Nuclear-Encoded Transcripts Identified in the E. chlorotica Transcriptome and Corresponding Sequence Matches in theAlgal Transcriptome and Genome Data.

Protein Identity (top BLAST species hit)a

NCBI Accession Number

Elysia chloroticaTranscript

IdentificationNumberb

Vaucheria litoreaTranscriptomeNCBI Accession

Numberc

V. litoreaGenomic Coding

SequenceNCBI Accession

Number

bp/bp Match(E. chlorotica/V. litoreasequence)

Phosphoribulokinase (V. litorea) AAK21910.1 44555290_d1 JP709200 JQ062392 90/9044555290_d2 JP709200 90/90

Light harvesting complex protein (V. litorea) ADD60136.1 33743048_d1 E52OB4B01DBNCL JQ062393 90/9033743048_d2 E52OB4B02I8050 90/90

Carbonic anhydrase (Ectocarpus siliculosus) CBN77745.1 5998831_d1 E52OB4B01BKQIY JQ062394 90/905998831_d2 JP709201 90/90

Carbonic anhydrase a type (E. siliculosus) CBN76519.1 23892389_d1 JP709202 JQ062395 90/9023892389_d2 E52OB4B02JG2P9 90/90

Carbonic anhydrase (Saccharina japonica) AEF33616.1 4706782_d1 JP709203 JQ062396 47/47d

4706782_d1 JP709203 90/90Pyrophosphate-dependent phosphofructokinase

(E. siliculosus) CBJ30811.122229246_d1 JP709204 JQ062397 88/9022229246_d2 JP709204 90/9022946887_d2 JP709204 90/90

Transketolase (E. siliculosus) CBJ48487.1 15424893_d1 JP709205 JQ062398 90/9015424893_d2 JP709205 90/9016607113_d1 JP709206 90/9016607113_d2 JP709206 89/90

Farnesyltransferase (Phaeodactylum tricornutum)XP_002178555.1

26003592_d1 JP709207 JQ062399 84/9026003592_d2 JP709207 90/90

Hsp-GrpE (E. siliculosus) CBN76646.1 20676998_d1 JP709208 JQ062400 89/9020676998_d2 JP709208 90/90

Chaperonin cpn60 (E. siliculosus) CBJ29911.1 17979526_d1 E52OB4B02GZ9VY JQ062401 90/9017979526_d2 E52OB4B02GZ9VY 90/90

Molecular chaperones Hsp70/Hsc70 Hsp superfamily(E. siliculosus) CBJ32839.1

19589706_d1 JP709209 JQ062402 90/9019589706_d2 JP709209 90/90

Soluble pyridine nucleotide transhydrogenase(E. siliculosus) CBN79739.1

26709754_d1 JP709210 JQ062403 84/84d

26709754_d2 JP709210 90/90Methyltransferase type 11 (Arthrospira maxima)

ZP_03275870.142534601_d1 JP709211 JQ062404 90/9042534601_d2 JP709211 90/90

Threonyl-tRNA synthetase (Phytophthora infestans)XP_002895146.1

43733749_d1 JP709296 JQ062405 90/9043733749_d2 JP709296 90/90

Rab8E.RAB family GTPase (E. siliculosus) CBN76228.1 32083728_d1 JP709212 JQ062406 89/9032083728_d2 JP709212 34/34d

Rab11A.RAB family GTPase (E. siliculosus) CBN78685.1 44639873_d1 E52OB4B01EGA6H JQ062407 90/9044639873_d2 E52OB4B01D56HY 90/90

ABC transporter-like protein (E. siliculosus) CBN74513.1 39105431_d1 JP709213 JQ062408 90/9039105431_d2 JP709213 90/90

ATPase (E. siliculosus) CBJ25903.1 5321251_d1 E52OB4B01BQB96 JQ062409 90/905321251_d2 None 87/90

Inorganic pyrophosphatase (E. siliculosus) CBJ29369.1 41485915_d1 E52OB4B01ES29L JQ062410 90/9041485915_d2 E52OB4B01ES29L 90/90

Trigger factor (TF) (E. siliculosus) CBJ32857.1 35301097_d1 JP709214 JQ062411 89/9035301097_d2 JP709214 89/90

Cellulase 2 (Pristionchus pacificus) ADV58320.1 26209836_d1 E52OB4B02IOHXW JQ062412 90/9026209836_d2 JP709297 85/85e

Tryptophan synthase (a/b chains) (E. siliculosus)CBN77109.1

38497509_d1 E52OB4B01AUQXK JQ062413 90/9038497509_d2 E52OB4B01AUQXK 88/90

Tocopherol polyprenyltransferase-like protein(Thalassiosira pseudonana) XP_002293015.1

45450736_d1 JP709215 JQ062414 89/9045450736_d2 JP709215 90/90

Ribosomal protein L3 (E. siliculosus) CBJ28620.1 14972294_d1 E52OB4B02GOMSI JQ062415 88/9014972294_d2 E52OB4B02GOMSI 90/90

Conserved unknown protein (E. siliculosus) CBN75765.1 600662_d1 JP709216 JQ062416 90/90600662_d2 JP709216 90/90

Conserved unknown protein (E. siliculosus) CBJ30937.1 20695943_d1 E52OB4B01BEOXI JQ062417 90/9020695943_d2 E52OB4B01BEOXI 89/90

Conserved unknown protein (E. siliculosus) CBJ33840.1 926198_d1 JP709217 JQ062418 90/90926198_d2 JP709217 90/90

Conserved unknown protein (E. siliculosus) CBJ29091.1 48792667_d1 JP709218 JQ062419 90/9048792667_d2 JP709218 90/90

Conserved unknown protein (E. siliculosus)CBJ28899.1

9456896_d1 E52OB4B01C4II9 JQ062420 90/909456896_d2 E52OB4B01C4II9 90/90


1552

Dow


ic.oup.com/m


ber 2021

Table 3Continued

Protein Identity (top BLAST species hit)a

NCBI Accession Number

Elysia chloroticaTranscript

IdentificationNumberb

Vaucheria litoreaTranscriptomeNCBI Accession

Numberc

V. litoreaGenomic Coding

SequenceNCBI Accession

Number

bp/bp Match(E. chlorotica/V. litoreasequence)


12058179_d1 JP709219 JQ062421 89/9012058179_d2 JP709219 89/90

Conserved unknown protein (E. siliculosus)CBN79086.1

13706224_d1 None JQ062422 90/9013706224_d2 E52OB4B02FYN72 90/90


24090625_d1 JP709220 JQ062423 90/9024090625_d2 JP709220 90/90


30948636_d1 JP709221 JQ062424 90/9030948636_d2 JP709221 90/9041324700_d1 JP709221 89/9041324700_d2 JP709221 90/90


19593353_d1 JP709222 JQ062425 89/9019593353_d2 JP709222 90/90


3907773_d1 E52OB4B01CTM1Z JQ062426 90/903907773_d2 E52OB4B02IV83X 90/90


12842227_d1 JP709223 JQ062427 90/9012842227_d2 JP709223 90/90


26886148_d1 JP709224 JQ062428 90/9026886148_d2 JP709224 90/90


48772180_d1 JP709225 JQ062429 90/9048772180_d2 JP709225 90/90

Expressed unknown protein (E. siliculosus)CBJ32627.1

20219847_d1 JP709226 JQ062430 90/9020219847_d2 JP709226 89/90

Putative sulfate permease family protein (E. siliculosus)CBN74518.1

6037510_d1 JP709227 JQ062431 90/906037510_d2 JP709227 90/90

Hypothetical protein Esi 0531 0003 (E. siliculosus)CBJ33624.1

8333052_d1 E52OB4B01BAHK0 JQ062432 90/908333052_d2 E52OB4B01BAHK0 87/9030302247_d1 E52OB4B01BAHK0 90/9030302247_d2 E52OB4B01BAHK0 86/90

Hypothetical protein Esi 0531 0003 (E. siliculosus)CBJ33624.1

42187880_d1 None JQ062433 90/9042187880_d2 None 90/90

Hypothetical protein THAPSDRAFT_22565(T. pseudonana) XP_002290531.1

19828103_d1 None JQ062434 90/9019828103_d2 None 90/90

Similar to CG7697-PA (E. siliculosus) CBN77571.1 15303709_d1 JP709228 JQ062435 82/83d

15303709_d2 JP709228 90/90Putative NIN-like transcription factor(E. siliculosus) CBN75385.1

22750097_d1 JP709298 JQ062436 90/9022750097_d2 E52OB4B01AL89S 89/90

Predicted protein (P. tricornutum)XP_002179782.1

21613523_d1 None JQ062437 90/9021613523_d2 None 90/90

No BLAST hit 6290510_d1 JP709229 JQ062438 88/906290510_d2 JP709229 90/90

No BLAST hit 36768416_d1 None JQ062439 90/9036768416_d2 None 44/44d

No BLAST hit 34498593_d1 E52OB4B02IHZHE JQ062440 90/9034498593_d2 E52OB4B02IHZHE 90/90

No BLAST hit 39685080_d1 None JQ062441 90/9039685080_d2 E52OB4B01CY5TH 90/90

No BLAST hit 16533926_d1 None JQ062442 26/26d

16533926_d2 None 89/90No BLAST hit 35063098_d1 E52OB4B02G8Q7G JQ062443 90/90

35063098_d2 E52OB4B02G8Q7G 90/90

a Cutoff 5 10�10.b All EC Illumina reads in this column are included in Sequence Read Archive Study number SRP009263.2. The labels in this column are identifiers used in supplementarytable 1, Supplementary Material online.c Singleton reads labeled E52OB4B0xxxxxx are included in Sequence Read Archive Study number SRP009267.2.d Fragment extended beyond the stop codon.e Fragment extended beyond the start codon.


1553

Dow


ic.oup.com/m


ber 2021




the synthesis of chlorophyll a by E. chlorotica (Pierce et al.2009). Of particular interest, chloroplast-encoded tran-scripts for a few chaperone and other protein-processingproteins were present in the slug RNA. This is the firsttime that any evidence for the synthesis of these kindsof chloroplast proteins has been found in a host cell,and they may provide clues to the underlying mecha-nisms of long-term plastid function in the foreign cyto-plasm.

Rarer than the chloroplast-encoded sequences in theE. chlorotica transcriptome were transcripts that matchedV. litorea nuclear-encoded genes for chloroplast proteins.All of these transcripts matched coding sequences inthe algal genome and most were present in one or bothof our algal transcriptome data sets. Annotation revealedgenes coding for Calvin cycle enzymes essential for the con-tinuation of photosynthesis, like algal specific sequencesfor prk, which had been found by PCR previously, as wellas for carbonic anhydrase, pfk and tkt. One of the V. litoreathylakoid membrane light harvesting components, lhc, aswell as thf1 (thylakoid formation protein) were also presentin the slug sequence data. Both of these, along with theother lhc’s found previously by PCR (Pierce et al. 2007;Schwartz et al. 2010) are important for the longevityand maintenance of the sequestered plastid. As was thecase with the chloroplast-encoded transcripts, several algalnuclear-encoded sequences for molecular chaperones orother protein-processing enzymes, including a trigger fac-tor homologue, which is involved in protein folding, ap-peared in the data. One of the more interesting aspectsof this intracellular symbiosis is the biochemical mechanismsthat integrate the chloroplast and host cell. While mostlyunknown at present, the mechanisms that prolong chloro-plast survival in the slug cells may rely on proteins such asthose.

Interestingly, 23 E. chlorotica transcripts that matchednuclear-encoded V. litorea sequences varied slightly fromthe native algal reference sequences by 1–6 bp. Nucleotidesubstitutions have also been observed in uroD (uropor-phyrinogen decarboxylase) located in slug cDNA andgDNA in earlier studies (Pierce et al. 2009). These differ-ences suggest that at least some of the transfer occurredsufficiently long ago to permit some of divergence to oc-cur between the native algal sequences and the corre-sponding sequences in the sea slugs.

Several other non–chloroplast-encoded sequences inthe E. chlorotica transcripts were annotated in the ‘‘con-served-‘‘, ‘‘unknown-,’’ or ‘‘hypothetical protein’’ categories.It is not presently possible to determine their functionalsignificance without more sequence information. However,almost all of those listed in those categories of our dataset had the E. siliculosus genome as the top BLASTspecies hit, indicating that they are most likely of algalorigin.

Thus, a variety of algal nuclear-encoded transcripts arepresent in the E. chlorotica transcriptome in spite of thetwo earlier reports to the contrary (Pelletreau et al.2011; Wagele et al. 2011). Several important issues need

to be considered to resolve these dramatic differences. First,different slug species were used by Wagele et al. (2011) and,except that it is indeed long standing, beyond the demon-stration of photosynthesis (Wagele and Johnsen 2001),almost nothing is known about the chloroplast symbiosisin P. ocellatus, including the algal species that donates thechloroplasts. Second, the P. ocellatus RNA was extractedfrom only a single specimen under undescribed conditionsthat may or may not have maximized the presence of tran-scripts for chloroplast proteins.

Third, a bit more is known about the chloroplast sym-biosis in the other species, E. timida, used by Wagele et al.(2011). Although it can last for several months, the chlo-roplast maintenance strategy of this species is behavioralshading of the chloroplast-residing body regions by meansof the parapodia, rather than any biochemical accommo-dations (Rahat 1976; Rahat andMonselise 1979; Casaldueroand Muniain 2008; Schmitt and Wagele 2011). Also, whilethese species are fairly distant from each other phylogenet-ically, in both, the anatomical location of the symbioticplastids is similar. Namely, they are mostly located in a dor-sal patch, where the digestive tubules rise to just below theepithelial surface between the two thick parapodial flaps,which can be extended over the chloroplast regions, effec-tively shading them from light (Wagele et al. 2011). With-out some sort of biochemical replacement, continualexposure to light will cause both the thylakoid compo-nents and photopigments to degrade. Indeed, degradedchloroplasts and rapid chlorophyll loss are evident instarved E. timida and parapodial shading behavior occursin response to light (Marın and Ros 1989, 1992). About10% of the symbiotic plastids are replaced over a 4-dayperiod if food is available, so E. timida seems to rely onfeeding and behavioral responses to sustain a fraction ofits symbiotic plastids long term and may not be an appro-priate species to test for horizontally transferred algal genes.Less is known with respect to P. ocellatus, but the anatomyand similar shading behavior suggest chloroplast protec-tion mechanisms similar to E. timida. In E. chlorotica, onthe other hand, the chloroplast containing ends of the di-gestive tubules underlie the epithelium over the entire sur-face of the slug, including the parapodia, so many of theplastids are in anatomical locations where shading is notpossible (http://www.seaslugforum.net/find/13756). Soamong the sacoglossans, more than one mechanism of plas-tid maintenance occurs (see also Pierce and Curtis 2012) andnot all may require algal genes, as in E. chlorotica.

Fourth, Wagele et al. (2011) assumed that, as in plantcells, there would be a very large chloroplast-targetedtranscript presence in the slug cells and thus, easily found.However, there is no basis for this assumption and, at leastin E. chlorotica, photosynthetic gene expression is muchslower than in the alga (Soule 2009).

Fifth, the symbiotic chloroplasts reside in only a few cellswithin the slugs. However, Wagele et al. (2011) extractedthe RNA from the entire animal in both slug species. Thus,almost all the RNA in their analyses came from cells that do


1554

Dow


ic.oup.com/m


ber 2021

http://www.seaslugforum.net/find/13756

not have chloroplasts and may not make transcripts forplastid proteins.

Sixth, in the case of both species, Wagele et al. (2011)examined only a small fraction of the transcriptomes andattempted to annotate them by comparison to the RefSeqdatabase, which contains few algal sequences and nonefrom Udoteacea, the algal taxon that seems to be the sourceof the symbiotic plastids. Not only did this process fail tofind any algal nuclear gene-like sequences, it returned onlyabout 6,000 BLAST hits from more than 77,000 assembledcontigs from the P. ocellatus data, for example, and of those,only 79 had a top homologue hit among plants. Of someimportance, 29 of these latter matched chloroplast-encodedgenes, indicating some plastid genome translational ac-tivity. The remaining 50 sequences matched genes ‘‘widelydistributed among eukaryotes.’’ The E. timida transcrip-tome analysis returned only two chloroplast-encoded genematches (Wagele et al. 2011). Perhaps more informationwould be found by comparison to data from species phy-logenetically closer to the actual chloroplast sources, espe-cially since sequence conservation between species amongseveral of the transferred algal nuclear genes found in E.chlorotica by PCR is low and do not hit anything in thegenomic data from Arabidopsis, Chlamydomonas, or Volvox(Pierce et al. 2007; Rumpho et al. 2008; Schwartz et al. 2010).So while the conclusion that neither P. ocellatus nor E. tim-ida contain transferred algal genes to support their symbi-otic chloroplasts may ultimately be correct, more evidencein addition to that presented by Wagele et al. (2011) seemsnecessary to establish that.

Finally, it is clear from our results that finding rare tran-scripts requires a relatively large sequencing effort andidentifying foreign sequences from phylogenetically distantspecies with little representation in the sequence databasesis facilitated by knowledge of native sequence. Sweepingconclusions based on small partial transcriptome analysescan be misguided. Like the other two sacoglossan studies,our RNA came from whole animal extracts, so most of itwas from cells that do not contain chloroplasts. In addition,the rarity of the transcripts in our data and the low rateof expression in the slug relative to the alga (Soule 2009;Pelletreau et al. 2011) suggest a low expression rate. Takentogether, a low expression rate by a relatively few cellswould conspire against finding the sequences in limitedamounts of data obtained from small amounts of RNA ex-tracted mostly from nonexpressing cells. Even our largeamount of data (8.8 Gbp) only returned a weak coverageof a few, mostly not overlapping, 90 bp fragments that didnot occur in sufficient number or variety to be formed intocontigs by the software. Indeed, the much smaller (148Mbp) E. chlorotica transcriptome data set was unsuccessfulat locating transferred genes (Pelletreau et al. 2011). Nodoubt, there are more transferred algal genes to be foundin the E. chlorotica DNA, which will require even moresequencing.

We are presently working on E. chlorotica genome se-quencing which may be the only way to get an estimateof the entire transferome. Although the initial discovery

of transferred genes in the slug genome was done withPCR techniques and, as a result of the specificity of PCR,returned only a few genes (Pierce et al. 2007; Rumpho et al.2008), the transcriptome analysis here indicates that manyalgal nuclear genes have somehow arrived into theE. chlorotica genome, and it is likely that others will befound. The size of the transferome suggests the possibilitythat pieces of DNA, rather than single genes, on multipleoccasions may be involved in the transfer and integrationprocess, but comparative genomics may provide theimportant answer to that.

Supplementary MaterialSupplementary tables 1 and 2 are available at MolecularBiology and Evolution online (http://www.mbe.oxfordjournals.org/).

AcknowledgmentsThe portion of the work done at the University of SouthFlorida was funded by a private donor, who wishes toremain anonymous. The portion of the work done atBGI-Shenzhen was done with funds provided by the BGIScience and Technology Foundation.

ReferencesAl-Samarrai TH, Schmid J. 2000. A simple method for extraction of

fungal genomic DNA. Lett Appl Microbiol. 30:53–56.Altschul SF, Gish W, Miller W, Myer EW, Lipman DJ. 1990. Basic local

alignment search tool. J Mol Biol. 215:403–410.Birney E, Clamp M, Durbin R. 2004. GeneWise and Genomewise.

Genome Res. 14:988–995.Casalduero FG, Muniain C. 2008. The role of kleptoplasts in the

survival rates of Elysia timida (Risso, 1818) (Sacoglossa:opisthobranchia) during periods of food shortage. J Mar BiolEcol. 357:181–187.

Darling A, Carey L, Feng W. 2003. The design, implementation, andevaluation of mpiBLAST. In: Proceedings of the ClusterWorldConference Expo and the 4th International Conference on LinuxCluster: The HPC Revolution; June, 2003; San Jose, CA.

Green BJ, Li W-Y, Manhart JW, Fox TC, Summer EJ, Kennedy RA,Pierce SK, Rumpho ME. 2000. Mollusc-algal chloroplastendosymbiosis. Photosynthesis, thylakoid protein maintenanceand chloroplast gene expression continue for many months inthe absence of the algal nucleus. Plant Physiol. 124:331–342.

Kent WJ. 2002. BLAT—the BLAST-like alignment tool. Genome Res.12:656–664.

Li R, Fan W, Tian G, et al. (116 co-authors). 2010. The sequence andde novo assembly of the giant Panda genome. Nature463(7279):311–317.

Li R, Zhu H, Ruan J, et al. (14 co-authors). 2010. De novo assembly ofhuman genomes with massively parallel short read sequencing.Genome Res. 20:265–272.

Lin H, Ma X, Chandramohan P, Geist A, Samatova N. 2005. Efficientdata access for parallel BLAST. In: Proceedings of 19th IEEEInternational Parallel and Distributed Processing Symposium;2005 Apr 3–8; Denver, CO. Los Alamitos (CA): IEEE ComputerSociety Conference Publishing Services.

Marın A, Ros J. 1989. The chloroplast-animal association of fourIberian Sacoglossan opisthobranchs: elysia timida, Elysia trans-lucens, Thuridilla hopei and Bosellia mimetica. In: Ros J, editor.


1555

Dow


ic.oup.com/m


ber 2021



http://www.mbe.oxfordjournals.org/

http://www.mbe.oxfordjournals.org/

Topics in marine biology. Proceedings of the 22nd EuropeanMarine Biology Symposium. Sci Mar. 53:429–440.

Marın A, Ros J. 1992. Dynamics of a peculiar plant–herbivorerelationship: the photosynthetic ascoglossan Elysia timida andthe chlorophycean Acetabularia acetabulum. Mar Biol. 112:677–682.

Mujer CV, Andrews DL, Manhart JR, Pierce SK, Rumpho ME. 1996.Chloroplast genes are expressed during intracellular symbioticassociation of Vaucheria litorea plastids with the sea slug Elysiachlorotica. Proc Nat Acad Sci. 93:12333–12338.

Pelletreau KN, Bharracharya D, Price DC, Worful JM, Moustafa A,Rumpho ME. 2011. Update on sea slug kleptoplasty and plastidmaintenance in a metazoan. Plant Physiol. 155:1561–1565.

Pierce SK, Biron RW, Rumpho ME. 1996. Endosymbiotic chloroplastsin molluscan cells contain proteins synthesized after plastidcapture. J Exp Biol. 199:2323–2330.

Pierce SK, Curtis NE. 2012. The cell biology of the chloroplastsymbiosis in sacoglossan sea slugs. Int Rev Cell Mol Biol. 293:123–148.

Pierce SK, Curtis NE, Hanten JJ, Boerner SL, Schwartz JA. 2007.Transfer, integration and expression of functional nuclear genesbetween multicellular species. Symbiosis 43:57–64.

Pierce SK, Curtis NE, Schwartz JA. 2009. Chlorophyll a synthesis byan animal using transferred algal nuclear genes. Symbiosis 49:121–131.

Rahat M. 1976. Direct development and symbiotic chloroplasts inElysia timida (Mollusca: Opisthobranchia). Israel J Zool. 25:186–193.

Rahat M, Monselise EBI. 1979. Photobiology of the chloroplast-hosting mollusc, Elysia timida (Opisthobranchia). J Exp Biol. 79:225–233.

Rumpho ME, Pochareddy S, Worful JM, Summer EJ, Bhattacharya D,Pelletreau KN, Tyler MS, Lee J, Marhart JR, Soule KM. 2009.Molecular characterization of the Calvin cycle enzyme phosphor-ibulokinase in the stramenopile alga Vaucheria litorea and theplastid hosting mollusc Elysia chlorotica. Mol Plant. 2:1384–1396.

Rumpho ME, Worful JM, Lee J, Kannan K, Tyler MS, Bhattacharya D,Moustafa A, Manhart JR. 2008. Horizontal gene transfer of thealgal nuclear gene psbO to the photosynthetic sea slug, Elysiachlorotica. Proc Natl Acad Sci U S A. 105:17867–17871.

Schmitt V, Wagele H. 2011. Behavioral adaptations in relation to long-term retention of endosymbiotic chloroplasts in the sea slug Elysiatimida (Opisthobranchia, Sacoglossa). Thalassas 27:225–238.

Schwartz JA, Curtis NE, Pierce SK. 2010. Using algal transcriptomesequences to identify transferred genes in the sea slug, Elysiachlorotica. Evol Biol. 37:29–37.

Soule KM. 2009. Light-regulated photosynthetic gene expressionand enzyme activity in the heterokont alga Vaucheria litorea andits symbiotic partner the sacoglossan mollusc, Elysia chlorotica[MS thesis]. [Orono (ME)]: University of Maine. p. 120.

Stanke M, Waack S. 2003. Gene prediction with a hidden Markovmodel and a new intron submodel. Bioinformatics 19(Suppl 2):ii215–ii225.

Wagele H, Deusch O, Handeler K, et al. (11 co authors). 2011.Transcriptomic evidence that longevity of acquired plastids inthe photosynthetic slugs Elysia timida and Plakobranchusocellatus does not entail lateral transfer of algal nuclear genes.Mol Biol Evol. 28:699–706.

Wagele H, Johnsen G. 2001. Observations on the histology andphotosynthetic performance of ‘‘solar-powered’’ opisthobranchs(Mollusca, Gastropoda, Opishobranchia) containing symbioticchloroplasts or zooxanthellae. Org Divers Evol. 1:193–210.


1556

Dow


ic.oup.com/m


ber 2021

Documents

Transcriptomic Evidence for the Expression of Horizontally